Although the depths of water uptake by plants can be estimated by comparing oxygen 18 or deuterium in stem water with that of possible water sources in semi-arid regions, it is difficult to apply this technique to investigations of water use in relatively humid areas because the vertical profile of the isotopic ratio in soil water in such areas is more complicated than that in arid and semi-arid regions. However, the d-excess (deuterium excess) of rainfall in Japan shows clear seasonal variations. Therefore, we attempted to utilize seasonal variations of d-excess to estimate water sources of 20-year-old Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Chriptomeria japonica). Source water could be distinguished based on differences in their seasonal pattern of d-excess. The seasonal variations of d-excess in the stem water of both species were similar to those of shallow soil water (<1.0 m), but different from those of other water sources, indicating that the main water source was shallow soil water.